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Novel cis-regulatory modules control expression of the Hairy and Enhancer of Split-1 (HES1) transcription factor in myoblasts.

Jeziorska DM, Koentges G, Vance KW - J. Biol. Chem. (2011)

Bottom Line: We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions.Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus.This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Systems Analysis, School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.

ABSTRACT
The expression profile of a gene is controlled by DNA sequences called cis-regulatory modules (CRMs). CRMs can function over large genomic distances and can be located many kilobases away from their target promoters. hes1 is a key developmental gene that is overexpressed in certain cancers and is a primary target of NOTCH signaling. Despite this, analysis of hes1 transcriptional control has been limited solely to its promoter. Here, we identify seven conserved DNA sequence blocks, representing the hes1 promoter and six novel CRMs, within 57 kb upstream of the mouse hes1 gene. We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions. We validate RBP-Jκ and TEAD family occupancy in cells in culture and test the response of each of these CRMs to active NOTCH. We show that two regions, CRM5 and CRM7, function as enhancers, and four can repress transcription. A pair of RBP-Jκ motifs arranged in a tail-tail configuration in CRM5 and the M-CAT motif in CRM7 are necessary for enhancer function. Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus. This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

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hes1 CRMs are bound by the RBP-JK and TEAD2 transcription factors.A, identification of RBP-JK motifs in the hes1 CRMs using the BiFa discovery tool. The position of each RBP-JK motif relative to the hes1 TSS is shown. Sequences matching the consensus high affinity PSSM are marked in gray and the architecture of the RPB-JK BSs within each CRM is indicated. Conserved BSs are marked by an asterisk. B, RBP-JK binds to hes1 CRMs in proliferating C2C12 cells. ChIP assays were performed using either an antibody against RBP-JK or an isotype-specific anti-GFP control (n = 4). The precipitated DNA fragments were PCR-amplified using primers spanning the predicted RBP-JK BSs within each CRM. The control represents an intervening region in the hes1 upstream sequence in which no RBP-JK BSs were detected in silico. Analysis using an unpaired Student's t test shows that RBP-JK occupancy at CRM1–6 is statistically significant compared with a nonbinding control region. * indicates p < 0.05; ** indicates p < 0.01. C, position of the conserved M-CAT motif in hes1 CRM7 and sequence matches to the consensus PSSM are shown. ChIP assays were performed in proliferating C2C12 cells using anti-TEAD1, anti-TEAD2, anti-TEAD4, or anti-GFP (isotype control) antibodies. Precipitated DNA fragments were PCR-amplified using primers spanning the predicted M-CAT motif in CRM7. To calculate specific enrichment over input, the signal for each PCR was quantified and divided by the input, and the background intensity, measured using an IgG isotope control, was subtracted. D, modulation of hes1 CRM-pro reporter activity by active NOTCH. Hes1CRM-reporter constructs were co-transfected into C2C12 cells along with a 1:1 ratio of reporter to NICD overexpression vector or an empty control vector to make the total amount of DNA transfected in each case equal. The results are presented as fold induction relative to the activity of each reporter in the absence of NICD and represent a mean ± S.D. of three independent experiments.
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Figure 3: hes1 CRMs are bound by the RBP-JK and TEAD2 transcription factors.A, identification of RBP-JK motifs in the hes1 CRMs using the BiFa discovery tool. The position of each RBP-JK motif relative to the hes1 TSS is shown. Sequences matching the consensus high affinity PSSM are marked in gray and the architecture of the RPB-JK BSs within each CRM is indicated. Conserved BSs are marked by an asterisk. B, RBP-JK binds to hes1 CRMs in proliferating C2C12 cells. ChIP assays were performed using either an antibody against RBP-JK or an isotype-specific anti-GFP control (n = 4). The precipitated DNA fragments were PCR-amplified using primers spanning the predicted RBP-JK BSs within each CRM. The control represents an intervening region in the hes1 upstream sequence in which no RBP-JK BSs were detected in silico. Analysis using an unpaired Student's t test shows that RBP-JK occupancy at CRM1–6 is statistically significant compared with a nonbinding control region. * indicates p < 0.05; ** indicates p < 0.01. C, position of the conserved M-CAT motif in hes1 CRM7 and sequence matches to the consensus PSSM are shown. ChIP assays were performed in proliferating C2C12 cells using anti-TEAD1, anti-TEAD2, anti-TEAD4, or anti-GFP (isotype control) antibodies. Precipitated DNA fragments were PCR-amplified using primers spanning the predicted M-CAT motif in CRM7. To calculate specific enrichment over input, the signal for each PCR was quantified and divided by the input, and the background intensity, measured using an IgG isotope control, was subtracted. D, modulation of hes1 CRM-pro reporter activity by active NOTCH. Hes1CRM-reporter constructs were co-transfected into C2C12 cells along with a 1:1 ratio of reporter to NICD overexpression vector or an empty control vector to make the total amount of DNA transfected in each case equal. The results are presented as fold induction relative to the activity of each reporter in the absence of NICD and represent a mean ± S.D. of three independent experiments.

Mentions: CRM function is mediated by the number and arrangement of different types of transcription factor- binding sites it contains. Given the important role played by NOTCH signaling in the control of hes1 expression, we analyzed the newly identified hes1 CRMs for RBP-JK BSs (Fig. 3A). RBP-JK is a major mediator of the NOTCH signaling pathway. To do this, we searched PSSMs from the TRANSFAC data base and calculated the likelihood of factor binding using BiFa, a custom discovery tool (30). This analysis identified the two known functional RBP-JK-binding sites in hes1 CRM1 validating our approach (7–9). Strikingly, the results also revealed an additional 10 high probability RBP-JK-binding sites within the newly identified mouse CRMs. The organization of these sites within each CRM as well as phylogenetic conservation and sequence similarity to the consensus high affinity PSSM is indicated in Fig. 3A. Our analysis did not predict any RBP-JK BSs with a high binding probability score in the intervening mouse genomic sequences. This suggests that these regions represent additional NOTCH-responsive transcriptional regulatory elements involved in the control of hes1 expression.


Novel cis-regulatory modules control expression of the Hairy and Enhancer of Split-1 (HES1) transcription factor in myoblasts.

Jeziorska DM, Koentges G, Vance KW - J. Biol. Chem. (2011)

hes1 CRMs are bound by the RBP-JK and TEAD2 transcription factors.A, identification of RBP-JK motifs in the hes1 CRMs using the BiFa discovery tool. The position of each RBP-JK motif relative to the hes1 TSS is shown. Sequences matching the consensus high affinity PSSM are marked in gray and the architecture of the RPB-JK BSs within each CRM is indicated. Conserved BSs are marked by an asterisk. B, RBP-JK binds to hes1 CRMs in proliferating C2C12 cells. ChIP assays were performed using either an antibody against RBP-JK or an isotype-specific anti-GFP control (n = 4). The precipitated DNA fragments were PCR-amplified using primers spanning the predicted RBP-JK BSs within each CRM. The control represents an intervening region in the hes1 upstream sequence in which no RBP-JK BSs were detected in silico. Analysis using an unpaired Student's t test shows that RBP-JK occupancy at CRM1–6 is statistically significant compared with a nonbinding control region. * indicates p < 0.05; ** indicates p < 0.01. C, position of the conserved M-CAT motif in hes1 CRM7 and sequence matches to the consensus PSSM are shown. ChIP assays were performed in proliferating C2C12 cells using anti-TEAD1, anti-TEAD2, anti-TEAD4, or anti-GFP (isotype control) antibodies. Precipitated DNA fragments were PCR-amplified using primers spanning the predicted M-CAT motif in CRM7. To calculate specific enrichment over input, the signal for each PCR was quantified and divided by the input, and the background intensity, measured using an IgG isotope control, was subtracted. D, modulation of hes1 CRM-pro reporter activity by active NOTCH. Hes1CRM-reporter constructs were co-transfected into C2C12 cells along with a 1:1 ratio of reporter to NICD overexpression vector or an empty control vector to make the total amount of DNA transfected in each case equal. The results are presented as fold induction relative to the activity of each reporter in the absence of NICD and represent a mean ± S.D. of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: hes1 CRMs are bound by the RBP-JK and TEAD2 transcription factors.A, identification of RBP-JK motifs in the hes1 CRMs using the BiFa discovery tool. The position of each RBP-JK motif relative to the hes1 TSS is shown. Sequences matching the consensus high affinity PSSM are marked in gray and the architecture of the RPB-JK BSs within each CRM is indicated. Conserved BSs are marked by an asterisk. B, RBP-JK binds to hes1 CRMs in proliferating C2C12 cells. ChIP assays were performed using either an antibody against RBP-JK or an isotype-specific anti-GFP control (n = 4). The precipitated DNA fragments were PCR-amplified using primers spanning the predicted RBP-JK BSs within each CRM. The control represents an intervening region in the hes1 upstream sequence in which no RBP-JK BSs were detected in silico. Analysis using an unpaired Student's t test shows that RBP-JK occupancy at CRM1–6 is statistically significant compared with a nonbinding control region. * indicates p < 0.05; ** indicates p < 0.01. C, position of the conserved M-CAT motif in hes1 CRM7 and sequence matches to the consensus PSSM are shown. ChIP assays were performed in proliferating C2C12 cells using anti-TEAD1, anti-TEAD2, anti-TEAD4, or anti-GFP (isotype control) antibodies. Precipitated DNA fragments were PCR-amplified using primers spanning the predicted M-CAT motif in CRM7. To calculate specific enrichment over input, the signal for each PCR was quantified and divided by the input, and the background intensity, measured using an IgG isotope control, was subtracted. D, modulation of hes1 CRM-pro reporter activity by active NOTCH. Hes1CRM-reporter constructs were co-transfected into C2C12 cells along with a 1:1 ratio of reporter to NICD overexpression vector or an empty control vector to make the total amount of DNA transfected in each case equal. The results are presented as fold induction relative to the activity of each reporter in the absence of NICD and represent a mean ± S.D. of three independent experiments.
Mentions: CRM function is mediated by the number and arrangement of different types of transcription factor- binding sites it contains. Given the important role played by NOTCH signaling in the control of hes1 expression, we analyzed the newly identified hes1 CRMs for RBP-JK BSs (Fig. 3A). RBP-JK is a major mediator of the NOTCH signaling pathway. To do this, we searched PSSMs from the TRANSFAC data base and calculated the likelihood of factor binding using BiFa, a custom discovery tool (30). This analysis identified the two known functional RBP-JK-binding sites in hes1 CRM1 validating our approach (7–9). Strikingly, the results also revealed an additional 10 high probability RBP-JK-binding sites within the newly identified mouse CRMs. The organization of these sites within each CRM as well as phylogenetic conservation and sequence similarity to the consensus high affinity PSSM is indicated in Fig. 3A. Our analysis did not predict any RBP-JK BSs with a high binding probability score in the intervening mouse genomic sequences. This suggests that these regions represent additional NOTCH-responsive transcriptional regulatory elements involved in the control of hes1 expression.

Bottom Line: We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions.Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus.This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Systems Analysis, School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.

ABSTRACT
The expression profile of a gene is controlled by DNA sequences called cis-regulatory modules (CRMs). CRMs can function over large genomic distances and can be located many kilobases away from their target promoters. hes1 is a key developmental gene that is overexpressed in certain cancers and is a primary target of NOTCH signaling. Despite this, analysis of hes1 transcriptional control has been limited solely to its promoter. Here, we identify seven conserved DNA sequence blocks, representing the hes1 promoter and six novel CRMs, within 57 kb upstream of the mouse hes1 gene. We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions. We validate RBP-Jκ and TEAD family occupancy in cells in culture and test the response of each of these CRMs to active NOTCH. We show that two regions, CRM5 and CRM7, function as enhancers, and four can repress transcription. A pair of RBP-Jκ motifs arranged in a tail-tail configuration in CRM5 and the M-CAT motif in CRM7 are necessary for enhancer function. Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus. This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

Show MeSH
Related in: MedlinePlus